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Plant and Soil 120, 195-201 (1989).
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PLSO 8184
Some properties of carbohydrate and C4-dicarboxylic acid utilization
negative mutants of Rhizobium leguminosarumbiovar phaseoli strain P121
P. J. LAFONTAINE, CAROLE LAFRENII~RE t and H. ANTOUN
D~partement des Sols, Facult~ des Sciences de l'Agriculture et de l'Alimentation, Universitk Laval, Qukbec,
Canada GIK 7P4. t present address." Station de recherche, Agriculture Canada, Kapuskasing, Ontario,
Canada P5N 2)(9
Received 20 March 1989. Revised July 1989
Key words:
french bean, oxygen consumption, Phaseolus vulgaris, Rhizobium leguminosarum biovar
phaseoli, symbiotic effectiveness
Abstract
After NTG treatment of the very effective wild type strain P121 of Rhizobium leguminosarum biovar
phaseoli, mutants defective in the utilization of sugars or organic acids were obtained. All the mutants
nodulated the cultivar Goldie of Phaseolus vulgaris. The arabinose, fructose, glucose and pyruvate utilization
mutants formed nodules similar in shape and size to the nodules formed by the wild type strain. These
mutants exhibited an acetylene reduction activity significantly lower than the activity observed with the wild
type strain. All the C4-dicarboxylic acid utilization mutants, formed ineffective nodules that did not show
a significant acetylene reduction activity. The C4-dicarboxylic acids uptake system is apparently inducible
in the free-living bacteria of strain P 121. When P121 cells were grown on glucose in the presence of 2.5 mM
malate, the rate of glucose-dependent 02 consumption significantly decreased suggesting the presence of a
catabolite repression-like phenomenon. Isolated bacteroids of strain P121, under the experimental conditions used, were able to oxidize succinate, fumarate or malate but did not oxidize pyruvate, glucose,
fructose or sucrose.
Introduction
of low 02 concentrations (Trinchant et al., 1981).
The present study shows that in Rhizobium
leguminosarum biovar phaseoli the ability to utilize
dicarboxylates is essential for the occurrence of
effective N2 fixation in French bean nodules, and
that the ability to utilize some other sugars is also
apparently important in this beneficial process, as
indicated by the significant alterations observed in
sugar utilization mutants.
In the Rhizobium-legumes symbiosis, the exchange of metabolites between plant and bacteroids play a crucial role in the establishment of an
effective nitrogen fixing system. Although the exact
nature of the carbon source supplied by the plant to
the bacteria is not known, bacteroids are believed
to receive primarily dicarboxylic acids (Emerich et
al., 1988). In fact, a functional C4-dicarboxylic acid
transport system is essential for N2 fixation to occur
in pea nodules (Finan et al., 1983), and the capacity
to utilize sugars is apparently not essential for bacteroid development or the establishment of effective N 2 fixation in pea (Glenn et al., 1984).
However, sucrose and glucose supported acetylene
reduction by bacteroids extracted from French
bean, soybean and pea root nodules in the presence
Material and methods
Bacter& and growth conditions
Rhizobium leguminosarum biovar phaseoli strain
PI 21, is a wild type strain very effective on Phaseolus vulgaris cv. Goldie (Lalande et al., 1986). Bac195
196
Lafontaine et al.
teria were grown as previously described
(Lafreni6re et al., 1987) on BMt or BM 2 media
supplemented with 0.5% sugars or 10mM organic
acids.
Isolation of mutant and revertant strains
Mutagenesis of strain P121 was carried out by
the treatment of the log phase cells grown on BM1mannitol, with 200#g/mL N-Methyl-N'-nitro-Nnitrosoguanidine (NTG) for 30 min at 25~ The
treated cells (20-40% survival) were washed twice
with phosphate buffer saline (PBS; 3mM
phosphate buffer in 0.7% NaC1, pH 6.8), suspended in BM~-mannitol broth and incubated for 24 h
at 28~ on a rotary shaker (160 rev.min J), to
allow segregation and expression of mutations. For
enrichment, the cells were washed twice in PBS and
resuspended in BM 2 supplemented with the
appropriate carbon source and 500/~g mL ~ carbenicillin were added. After incubation at 28~ for
24 h on a rotary shaker, the enrichment cycle was
repeated. Enriched cultures were washed and
plated for single colonies on BM2 medium modified
(Lafreni6re et al., 1987) by using 0.6 g L l proteose
peptone (Difco) as a nitrogen source and by adding
filter sterilized 2,3,5-triphenyl tetrazolium chloride
(TTC, Sigma) to a final concentration of
25 mg L- i. On this medium wild type colonies have
a good growth and reduce TTC (pink colonies),
while mutants show poor growth and TTC is not
reduced (white colonies). Revertants were selected
by plating a dense washed culture on BM2succinate plates. Mutants and revertants were
preserved at - 80~ in yeast extract mannitol broth
containing 10% glycerol.
Plant growth conditions
French bean (Phaseolus vulgaris cv. Goldie)
plants were grown in an autoclaved hydroponic
growth system as previously described (Lafontaine
et al., 1989).
22 days after plants inoculation by incubating for
30 min, the 2 detached root systems obtained from
each hydroponic jar, in a 150mL glass bottle,
capped with Subba Seal (William Freeman Co.,
Barnsley, England), and containing 10% acetylene.
The ethylene produced was measured with a
Perkin Elmer Sigma 3B dual FID gas chromatograph equipped with a 1 m stainless steel column
packed with porapack R (80-100 mesh) and
operated at 45~ The injector and detector temperatures were maintained at 55~ and 125~ respectively.
Bacteroids isolation
Phaseolus vulgaris cv. Goldie nodules were
removed from the roots and gently crushed in a
solution of 0.15 M NaC1 and 50 mM KH2PO4, pH
7.6 at 4~ This crude homogenate was filtered
through 6 layers of cheesecloth, and l mL was
layered on top of a 70% Percoll solution (Reibach
et al., 1981) and centrifuged at 40, 360 • g for
40 min at 4~ in an IEC 33~ angle head rotor (IEC
model B-20 refrigerated centrifuge). The cytosol
fraction, on top of the Percoll gradient and the
bacteroids fraction near the bottom of the
centrifuge tube were collected with Pasteur pipettes. Percoll was removed by diluting the bacteroid
fraction 1:5 (v/v) with 0.15 M NaC1 plus 50mM
KHzPO4 pH 7.6 and centrifuging 10min at
12000 • g. The bacteroid pellet was resuspended
in BM1 without carbon source.
Measurement of 02 consumption
Substrates dependent 02 consumption by freeliving bacteria and bacteroids was measured by
using a biological oxygen monitor (Yellow Spring
Instrument, Ohio) as previously described
(Lafreni6re et al., 1987).
Protein determination
Acetylene reduction activity
The acetylene reduction activity was measured
For protein determination cells were washed in
PBS and digested at 90~ in l M NaOH for 10 min
and the protein content was determined by the
Properties of R. leguminosarum bv phaseoli mutants
Folin phenol method (Lowry et al., 1952) using
bovine serum albumin as a standard.
Results and discussion
Isolation and growth properties of carbohydrate
and organic acid-utilization mutants
Strain P121 is a very effective strain of R.
leguminosarum biovar phaseoli, isolated from a
Quebec soil (Lalande et al., 1986). This strain grows
on sugars and organic acids commonly used by
rhizobia. After NTG mutagenesis and carbenicillin
enrichment, the carbohydrate (arabinose, P 121A 18;
glucose, PI21DH; fructose, P121FH1 and FH2)
mutants isolated, were able to use organic acids and
other carbohydrates as sole carbon source (lable
1). Pyruvate was the only carbon source not used
by mutant P121P22. All C4-dicarboxylic acid
mutants (P121 $4, P121S9 and P121S21) did not use
succinate, fumarate and malate as sole carbon
source, but they were able to grow on glucose and
arabinose indicating that they have a functional
tricarboxylic acid cycle (Bolton et al., 1986;
Duncan and Fraenkel, 1979). Thus, the failure of a
mutant to utilize succinate, fumarate and malate
was taken as an indication of a defect in the C4Table 1.
Growth
Strain
of
R. leguminosarum
Carbon
biovar
phaseoli
dicarboxylic acid transport system, recognized to
be common for the three organic acids (Finan et al.,
1981). Strain P121F16 is a double mutant defective
in the utilization of dicarboxylic acids and fructose,
but able to grow on glucose and arabinose (Table
1). Strains P121S21R10 and P121S21RI4 are revertants of the mutant strain P121S21 selected on
succinate. These revertants grew on all carbon
sources used by the wild type strain P121.
Symbiotic properties of mutant and revertant
strains
All mutant and revertant strains nodulated
French bean (Phaseolus vulgaris cv. Goldie).
The carbohydrate utilization mutants P121FHI,
P121FH2 and P121DH, the pyruvate mutant
P121 P22 and the revertant strains P 121 $21 R 10 and
P 121 $21 R 14, formed effective pink nodules similar
in shape, size and number to those formed by the
wild type strain Pi21. The arabinose utilization
mutant P 121A 18 also formed effective nodules but
in lesser number. The C4-dicarboxylic acid utilization mutants P121S4, P121S9 and P121S21 and the
double mutant P121F16 nodulated French bean,
but the nodules were ineffective, small and white to
greenish.
P 1 2 1 a n d its m u t a n t s
and revertants on some carbon sources
source
Pyruvate
Succinate
Fumarate
Malate
Glucose
Fructose
Arabinose
+
+
+
+
+
+
+
Wild type
PI21
197
Mutants
Carbohydrates
PI21A18
+
+
+
+
+
+
-
PI21DH
+
+
+
+
-
+
+
P121FH1
+
+
+
+
+
-
+
P121FH2
+
+
+
+
+
-
+
Organic acids
P121F16
+
-
-
-
+
-
+
P121P22
-
+
+
+
+
+
+
PI21S4
+
-
-
-
+
+
+
P121S9
+
-
-
-
+
+
+
P121S21
+
-
-
-
+
+
+
P121S21R10
+
+
+
+
+
+
+
P121S21RI4
+
+
+
+
+
+
+
Revertants
198
Lafontaine et al.
All the carbohydrate utilization mutants
retained their ability to fix dinitrogen as indicated
by the presence of an acetylene reduction activity
(Table 2). This suggests as previously reported for
the R. leguminosarum biovar viceae (Glenn et al.,
1984) that the capacity to utilize some sugars is
apparently not essential for bacteroid development
or the establishment of effective N2 fixation.
However, the nitrogenase activity detected in
French bean plants nodulated with the carbohydrate utilization mutants was always significantly lower than that observed in plants
nodulated with the wild type strain P121 (Table 2).
The lowest nitrogenase activity observed with the
arabinose utilization mutant P121A18 (32% of
P121 activity) could be attributed in part, to the low
number of nodules formed. The acetylene reduction activities observed with the other mutants
ranged from 47 to 78% that of the wild type strain
P121, with the pyruvate (P121P22) and the glucose
(P121DH) utilization mutants respectively. As the
nodulation of these mutants was similar to the
nodulation of the wild type effective strain, this
might indicate that the capacity to utilize glucose,
fructose or pyruvate by R. leguminosarum biovar
phaseoli bacteroids, is in part essential for an
optimum nitrogenase activity. In fact, sucrose and
glucose supported acetylene reduction by bacteroids extracted from French bean root nodules in
the presence of low 02 concentrations but not
under 02 tensions usually able to support acetylene
reduction with succinate (Trinchant et al., 1981).
All the mutants that are altered in the utilization
of succinate, fumarate and malate, including the
double mutant P121FI6, have lost completely their
ability to fix N2. The acetylene reduction activities
observed with these mutants are not statistically
different from the activity observed with the
uninoculated plants (Table 2). The results shown,
corroborate previous observations (Arwas et al.,
1985; Finan et al., 1983) indicating that utilization
of exogenous dicarboxylates (i.e. possession of a
functional C4-dicarboxylic acid transport system)
is essential for N 2 fixation to occur in nodules.
However, all carbohydrate mutants had altered
nitrogenase
activity
and
one
revertant
(P 121 $21R 10) had a restored nitrogenase activity
which is also significantly lower than that of the
Table 2. Nodulation and acetylene reduction activity (#moles C2 H4 h-E p l a n t - t ) of Phaseolus vulgaris cv. Goldie inoculated with R.
leguminosarum biovar phaseoli P121 and its m u t a n t and revertant strains
Strain
Nodulation
Acetylene
reduction
activity
Wild type
PI21
+
6.44 a a
Carbohydrates
PI21AI8
P121FH1
PI21FH2
P121DH
+
+
+
+
2.06
3.72
4.72
5.04
d
cd
bcd
b
Organic acids
P121F16
P121P22
P121S4
P121S9
P121521
+
+
+
+
+
0.12
3.05
0.89
0.29
0.I0
e
cd
e
e
e
Revertants
P121S21R10
PI21S21R14
+
+
4.69 bc
5.22 ab
Uninoculated
--
0.09 e
Mutants
a Means followed by the same letter are not significantly different (P ~< 0.05) according to the Waller D u n c a n ' s multiple range test.
Means are from 3 replicates.
Properties of R. leguminosarum by phaseoli mutants
effective wild type strain (Table 2). This suggests
that R. leguminosarum biovar phaseoli bacteroids
are using more than one carbon source, to establish
an effective nitrogenase activity in Phaseolus vulgaris nodules. The ability of the double mutant
P121F16 to nodulate French bean also suggests
that R. leguminosarum biovar phaseoli can use
carbon sources other than C4-dicarboxylic acids or
sugars to fuel the nodulation process. Similar observations were previously reported with mutants
of R. leguminosarum biovar viceae (Arwas et al.,
1986).
Oxidation of substrates by R. leguminosarum
biovar phaseoli P121 and its C4-dicarboxylic acid
utilization mutant P121S21
Because of the importance of the role played by
dicarboxylates in the symbiotic nitrogen fixation by
French bean nodules, we have compared the oxidation of malate and glucose by the effective wild type
strain P121 and its ineffective C4-dicarboxylic acid
utilization mutant strain P121S21.
R. leguminosarum biovar phaseoli PI21 cells
grown on glucose were not able to oxidize readily
pyruvate, succinate, malate and fumarate (Table 3).
However, when grown on malate P121 was capable
of oxidizing the three organic acids succinate,
malate and fumarate but pyruvate was not
oxidized, and the glucose and fructose-dependent
02 consumptions significantly decreased. These observations show that as previously reported for
Rhizobium leguminosarum biovar viceae (Finan et
al., 1981; Glenn et al., 1980), R. leguminosarum
biovar phaseoli P121 possesses a C4-dicarboxylic
acid uptake system which is inducible and mediates
the uptake of succinate, malate and fumarate. The
199
absence or very low pyruvate, glucose and fructosedependent 02 consumptions suggests, that strain
PI21 has an inducible oxidation system for the
catabolism of these carbon sources, or the presence
of a catabolite repression-like phenomenon. The
systems for the catabolism of pyruvate, glucose and
fructose are constitutive in a strain of Rhizobium
leguminosarum biovar viceae (Glenn and Dilworth,
1981). Regardless of the carbon source used in the
cell culture medium, strain P121 $21 was not able to
oxidize succinate, malate, fumarate or pyruvate but
it oxidized glucose and fructose (Table 3).
No significant organic acid-dependent 02 consumption was observed with PI21 cells grown on
glucose. The presence of a catabolite repressionlike phenomenon (Lafreni6re et al., 1987) was investigated by growing strain P 121 in BM2 mediu m
containing glucose and an increasing concentration
of malate. The addition of 2.5 mM or more malate
significantly decreased the rate of glucose-dependent 02 consumption (Table 4), indicating the presence of a catabolite repression-like phenomenon
in strain P121. In fact, such a repression is absent
in the C4-dicarboxylic acid utilization mutant
P121 $21 (Table 3). The presence of the lowest concentration of malate used, was also necessary for
the occurrence of a significant malate-dependent 02
consumption (Table 4), which confirm the presence
of an inducible C4-dicarboxylic acid uptake system
in strain P121.
Oxidation of substrates by bacteroids of strain
P121 and its C4-dicarboxylic acid utilization
mutant P121S21
The isolated bacteroids of strain P121, exhibited
a significant substrate-dependent 02 consumption
Table 3. Substrate-dependent 02 consumption by free-living cells of R. leguminosarum biovar phaseoli P 121 and its C4-dicarboxylic acid
utilization mutant P121S21
Carbon source
in growth
medium
Strain
Malate
(I0 mM)
P121
P121S21 ~
Glucose
(0.5%)
PI21
Pt21S21
Rate of 02 consumption in nmoles O, min- ~ (mg protein)- ~ in the presence of:
Pyruvate
(10 mM)
Succinate
(10mM)
Fumarate
(10 raM)
Malate
(10mM)
Glucose
(0.5%)
Fructose
(0.5%)
11.99
0.00
375.15
1.85
238,62
20,30
295.20
0.00
23.78
125.54
39.69
115.41
9.43
6.97
5.33
4.51
7,79
020
11.77
11.89
180.81
135.72
211.56
19434
a Mutant was grown on BM 2 with 0.5% glucose and transferred on BM 2 with 10 mM malate for 15 h prior to 0 2 measurements.
200
Lafontaine et al.
Table 4. Substrate-dependent 0 2 consumption by flee-living
cells of R. leguminosarum biovar phaseoli PI21 grown on BM 2
medium containing 0.5% glucose and supplemented with
increasing concentrations of malate
Concentration
of malate
(mM)
0.0
2.5
5.0
10.0
Table 5. Substrate-dependent 02 consumption by isolated bacteroids of R. leguminosarum biovar phaseoli P121 and its C4dicarboxylic acid utilization mutant P I 21 $21
Substrate
Concentration
Rate of 0 2 consumption in nmoles 0 2
rain 1 (mg protein)-~ in the presence
of:
Rate of 02 consumption in nmoles
02 min ~ (rag
protein) J
Glucose (0.5%)
Malate (10mM)
PI21
P121S21
137.60
32.80
23.78
12.71
14.76
136.65
144.74
137.60
0.9
3.3
68.9
32.6
47.8
1.4
0.8
0.3
0.5
1.8
1.3
0.6
0.9
0.8
0.9
0.7
52.8
1.1
only with the organic acids succinate, malate and
fumarate (Table 5). The bacteroids of the ineffective C4-dicarboxylic acid utilization mutant
P121S21 did not oxidize any of the carbon sources
tested. The respective plant cytosol fractions,
stimulated the respiration of P121 bacteroids but
had not any effect on P121S21 bacteroids. Our
results are similar to other observations made with
bacteroids of a strain of R. leguminosarum biovar
viceae (Glenn and Dilworth, 1981) and a strain of
cowpea Rhizobium (Saroso et al., 1984).
The results presented here indicate that C4-dicarboxylic acid utilization by bacteroids is essential for
the establishment of an effective Rhizobium
legurninosarum bv. phaseoli - - Phaseolus vulgaris
symbiosis. This supports the previous hypothesis
(Ronson et al., 1981) which states that C4-dicarboxylic acids are the main source of energy and
reducing power for N2-fixing bacteroids. In
Phaseolus vulgaris nodules, a high concentration of
malate can be detected (Lafontaine et al., 1989;
Streeter, 1987). Moreover, in ineffective nodules
induced by strain P121S21, the concentration of
malate was tenfold higher than in effective P121induced nodules (Lafontaine et al., 1989). Thus, the
possibility of the existence of a catabolic
repression-like phenomenon mediated by malate
and blocking the catabolism of glucose in bacteroids should be investigated. Reports on the
support of acetylene reduction by glucose in
French bean under low 02 concentrations (Trinchant et al., 1981) and the significant alterations of
the nitrogenase activity in some sugar utilization
negative mutants, are points in favor of the
repression hypothesis. More studies on the transport of metabolites across the peribacteroid membrane under different conditions, and an exhaustive
Pyruvate
6-P-Gluconate
Succinate
Fumarate
Malate
Glucose
Fructose
Sucrose
10 mM
5 mM
10 mM
10 mM
10 mM
0.5%
0.5%
0.5%
Cytosol
biochemical characterization of the carbohydrate
mutants obtained in this study, are essential to
elucidate the exact nature of the carbon source
supplied to the nitrogen fixing bacteroids.
Acknowledgments
This work was supported by a grant from the
Natural Sciences and Engineering Research
Council of Canada (NSERC). Cost of the present
paper is supported by the Conseil de recherches et
services agricoles du Qu6bec.
References
Arwas R, McKay I A, Rowney F R P, Dilworth M J and Glenn
A R 1985 Properties of organic acid utilization mutants of
Rhizobium leguminosarum strain 300. J. Gen. Microbiol. 13I,
2059-2066.
Arwas R, Glenn A R, McKay I A and Dilworth M J 1986
Properties of double mutants of Rhizobium leguminosarum
which are defective in the utilization of dicarboxylic acids and
sugars. J. Gen Microbiol. 132, 2743-2747.
Bolton E, Higgison B, Harrington A and O'Gara F 1986 Dicarboxylic acid transport in Rhizobiurn meliloti: Isolation of
mutants and cloning of dicarboxylic acid transport genes.
Arch Microbiol. 144. 142-146.
Duncan M J and Fraenkel D G 1979 ~-Ketoglutarate
dehydrogenase mutant of Rhizobium meliloti. J. Bacteriol. 37,
415-419.
Emerich D W, Anthon G E, Hayes R, R, Karr D B, Liang R,
Preston G G, Smith M T and Waters J K 1988 Metabolism
of Rhizobium-leguminous plant nodules with an emphasis on
Properties of R. leguminosarum by phaseoli mutants
bacteroid carbon metabolism. In Nitrogen Fixation: Hundred
Years After; Proc. 7th Intern. Congress on N 2 Fixation, K61n.
Eds. H Bothe, F J de Bruijn and W E Newton. pp 539-546.
Gustav Fischer Publishers, Stuttgart.
Finan T M, Wood J M and Jordan D C 1981 Succinate transport in Rhizobium leguminosarum. J. Bacteriol. 148, 193-202.
Finan T M, Wood J M and Jordan D C 1983 Symbiotic properties of C4-dicarboxylic acid transport mutants of Rhizobium
legurninosarum. J. Bacteriol. 154, 1403-1413.
Glenn A R and Dilworth J 1981 Oxidation of substrates by
isolated bacteroids and free-living cells of Rhizobium
leguminosarum 3841. J. Gen. Microbiol. 126, 243-247.
Glenn A R, Poole P S and Hudman J F 1980 Succinate uptake
by free-living and bacteroids forms of Rhizobium
leguminosarum. J. Gen. Microbiol. 119, 267-271.
Glenn A R, McKay I A, Arwas R and Dilworth M J 1984 Sugar
metabolism and the symbiotic properties of carbohydrate
mutants ofRhizobium legurninosarum. J. Gen. Microbiol. 130,
239-245.
Lafontaine P J, Lafreni+re C, Chalifour F-P, Dion P and
Antoun H 1989 Carbohydrate and organic acid composition
of effective and ineffective root nodules of Phaseolus vulgaris
L. Physiol. Plant. (In press).
Lafreni~re C, Lafontaine P, Marion C and Antoun H 1987
Oxidation of substrates in organic acids utilization mutants
and the wild type Rhizobium rneliloti strain SI4. Plant and Soil
201
101, 73-78.
Lalande R, Antoun H, Par6 T and Joyal P 1986 Effets de
l'inoculation avec des souches du Rhizobium leguminosarum
biovar phaseoli sur le rendement et la teneur en azote du
haricot (Phaseolus vulgaris). Naturaliste Can. 113, 337-346.
Lowry O, Rosembrough N, Farr A and Randall R 1952 Protein
measurement with the Folin phenol reagent. J. Biol. Chem.
193, 265-275.
Reibach P H, Mask P L and Streeter J G 1981 A rapid one-step
method for the isolation of bacteroids from root nodules of
soybean plants, utilizing self-generating Percoll gradients.
Can. J. Microbiol. 27, 491-495.
Ronson C W, Littleton P and Robertson J G 1981 C4-dicarboxylate transport mutants of Rhizobium trifolii form ineffective nodules on Trifolium repens. Proc. Natl. Acad. Sci. USA.
78, 4284-4288.
Saroso S, Glenn A R and Dilworth M J 1984 Carbon utilization
by free-living and bacteroid forms of cowpea Rhizobium
strain NGR234. J. Gen. Microbiol. 130, 1809-1814.
Streeter J G 1987 Effect of nitrate on the organic acid and amino
acid composition of legume nodules. Plant Physiol. 85, 774-779.
Trinchant J C, Birot A M and Rigaud J 1981 Oxygen supply and
energy-yielding substrates for nitrogen fixation (acetylene
reduction) by bacteroids preparations. J. Gen. Microbiol.
125, 159-165.